PROJECT SUMMARY The majority of new human diseases arise from zoonotically transmitted viruses, yet most research on cross-species transmission is focused on a few model RNA viruses. There is a fundamental gap in understanding how DNA viruses rapidly adapt to infect new hosts in spite of their relatively high replication fidelity. Our long- term goal is to define molecular and evolutionary signatures that identify ?high-risk? viruses poised to cross- species barriers before an outbreak occurs. As a step towards this goal, our current objective is to define how an experimentally evolved vaccinia virus (VACV) adapted to evade host antiviral proteins in a species-specific manner. Our central hypothesis is that initial adaptation by gene duplication acts as a ?molecular foothold?, to broadly improve viral replication in resistant host species, and thereby enable subsequent mutations to emerge that allow collapse of the amplified locus while maintaining the fitness benefit needed for continued replication and spread in the new host. This hypothesis is based on our work using a cell culture-based system of experi- mental evolution demonstrating that gene amplification of a weak viral antagonist can rapidly improve virus rep- lication in otherwise resistant cells derived from multiple species. Subsequent adaptive mutations evolved in the viral RNA polymerase (vRNAP) that differentially rescue virus replication in cells derived from multiple primate species. The rationale underlying the proposed research is that once it is known how these mutations inhibit immune proteins, we can more precisely identify poxviruses that are a higher risk to establish productive human infections. This knowledge will also inform strategies to design safer poxvirus-vectored vaccines and oncolytic agents that are less likely to evolve and become more pathogenic in patients. We plan to test our central hypoth- esis for this project by completing the following two specific aims: 1) Determine how experimentally evolved mutations in VACV vRNAP alter transcription and translation to inhibit dsRNA-mediated host restriction factors. We will use pulse-chase RNASeq methods to define transcriptome-wide changes in the kinetics of RNA synthe- sis and dsRNA accumulation in wt and vRNAP mutant viruses. We will also use high-dynamic range mass- spectrometry techniques to identify changes in the viral proteome due to these mutations. 2) Define how AGM- adapted viruses evolved to efficiently replicate in human fibroblasts. We will use our established genomics pipe- line and newly available single molecule sequencing to define adaptive mutations that evolved during serial passage in HF and identify structural variation in these viruses that may facilitate rapid adaptation of DNA viruses. We believe that the research proposed in this application is innovative because it represents a novel departure from most current viral cross-species transmission research by shifting focus to defining molecular and evolu- tionary signatures of DNA viruses undergoing the process of rapid adaptation. The proposed research is signif- icant because it will provide fundamental insights into DNA virus evolution and vRNAP biology that will open new avenues of research investigating transcriptional and translational changes during cross-species adaptation.